Our long term goals are to understand the biosynthesis, regulation and function of the carbohydrate portion of membrane glycoproteins. Since N-linked and O-linked oligosaccharides are found as components of many membrane receptors, such as those for low density lipoprotein, insulin and acetylcholine, these studies have important implications in a variety of disease conditions such as atherosclerosis and diabetes. Our strategy to achieve these goals will involve a multifaceted approach. We will purify from pig aorta several key enzymes that we think, and the literature suggests, are likely to be regulatory enzymes in the biosynthesis of N-linked oligosaccharides. After purification to homogeneity, antibody will be made against the proteins and oligonucleotide probes will be synthesized based on the amino-terminal or other peptide sequences. These tools will enable us to determine the levels of these enzymes and their mRNAs at various stages of growth or development, and in normal animals or animals fed high cholesterol diets. If the proteins in aortic smooth muscle cells cross react with the antibodies, levels of enzymes will be studied in these cells treated with various inhibitors. These studies will be correlated with effects of inhibitors on the formation of lipid-linked oligosaccharides in cultured smooth muscle cells. Another approach will be to use inhibitors of N-linked and O-linked oligosaccharide biosynthesis to study formation and function. We have several new inhibitors of processing of N-linked oligosaccharides that we want to characterize since they have new inhibitory properties and will provide important information about structure-inhibition relations. At present there are no useful inhibitors of O-linked oligosaccharides or glycosyl transferases. Thus, we will develop some rapid methods to look for such inhibitors and will test a number of antibodies and other glycosides that we have obtained from various sources. Finally, we have compelling evidence for the presence of mannosyl-O-serine linkages in several different animal cell lines grown in culture. We will determine the optimum conditions for introducing labeled mannose into these proteins and then do more detailed characterization to establish this linkage. The protein(s) having the mannosyl-serine will be purified to homogeneity so that we can prepare antibody and oligonucleotide probes in order to establish the location and function of the protein. The biosynthesis of the mannosyl-serine will be studied and the mannosyl donor determined.